The present invention relates generally to immunoassays for analyte detection and more particularly to immunoassays which rely on a change in a signal to detect analyte concentrations which exceed a preselected threshold value.
Immunoassays which produce a visual signal, such as color change, fluorescence, luminescence, or the like, are particularly useful for the detection of threshold concentrations of an analyte. Desirably, such immunoassay systems will provide an extreme signal condition when the concentration of analyte in a sample is below the preselected concentration value and will provide the opposite extreme signal condition when the analyte concentration exceeds the concentration value even by a small amount.
Of particular interest to the present invention are membrane assays of the type described in U.S. Pat. No. 4,818,677, and lateral flow assays of the type described in U.S. Pat. No. 4,943,522, the disclosures of which are incorporated herein by reference. Certain membrane assays utilize a reaction cell having a microporous membrane placed over an absorbant capable of drawing a liquid sample perpendiculary through the membrane. A capture reagent, typically an antibody, specific for the analyte is immobilized on the membrane. Sample which is applied to the membrane is drawn therethrough by the absorbant, with any analyte present being captured by the reagent. Signal can be produced by further applying labelled antibodies or other reagents which specifically recognize the analyte and mediate the production of a signal, such as color, fluorescence, or luminescence.
Lateral flow assays also utilize a porous membrane for performing a detection reaction. Instead of drawing the sample through the membrane perpendicularly, however, the sample is permitted to flow laterally from an application zone to a reaction zone on the membrane surface. The capture reagent is present in the reaction zone, and the captured analyte can be detected by a variety of protocols, including direct visualization of visible moieties associated with the captured analyte (as described in copending application Ser. No. 07/639,967, the disclosure of which is incorporated herein by reference).
By employing antibodies having a high affinity for the analyte as the capture reagent, a steep or rapid change in the visual signal can be achieved. That is, the signal will change over a very narrow concentration band. While such a rapid or sharp change is desirable, it is also necessary that the change occur at the concentration value of interest. If the signal change occurs above or below the concentration value of interest, the information provided by the assay will be of reduced value.
One approach for altering the detected concentration value is described in U.S. Pat. No. 4,952,517, and European Patent Appln. No. 327 843. A sample having an unknown amount of analyte is combined with a preselected threshold amount of free anti-analyte. The anti-analyte binds the analyte, with unbound anti-analyte remaining only if the original analyte concentration in the sample was below the threshold value (determined by the preselected amount of free anti-analyte). The sample is then contacted with a limited amount of immobilized analyte which will bind to free anti-analyte remaining in the sample. By then exposing the immobilized analyte to labelled anti-analyte, label will bind only if the analyte concentration in the sample was below the threshold concentration. The bound label can then be detected by a variety of conventional techniques to produce a visual signal. Thus, by properly selecting the concentration of free anti-analyte added to the sample and the amount of immobilized analyte, both the concentration detected and the range over which the signal changes from minimum to maximum can be controlled.
While generally workable, the sensitivity of this method is generally limited by the amount of immobilized analyte reacted with the sample (i.e., the method is not suitable for detecting threshold amounts lower than the amount of immobilized analyte). Moreover, high analyte concentrations in the sample can interfere with the results of the assay. That is, excessive amounts of free analyte could be carried over and bind the labelled anti-analyte, affecting the result. Additionally, the concentration range of analyte is limited by the amount of immobilized analyte.
A second approach for performing visual signal immunoassays for detecting preselected concentration values is described in U.S. Pat. No. 5,028,535, and corresponding PCT application WO 90/08319. A sample containing an unknown amount of native analyte is contacted with known amounts of labelled analyte and analyte receptor, usually anti-analyte antibody, to form a reaction mixture. Competition between the labelled analyte and the unknown (unlabelled) analyte for binding to the analyte receptor occurs, with the amount of labelled analyte remaining unbound being directly proportional to the amount of unknown analyte originally present in the sample. After separation, the amount of unbound labelled analyte can be detected by conventional visual detection systems and related directly to the amount of analyte initially present in the sample.
While generally workable, these detection systems rely on competition between native analyte and labelled analyte, requiring time to reach equilibrium. Moreover, the sensitivity of the assay may be limited by the amount of analyte receptor and the time delay in reaching equilibrium. Additionally, the assay system may be influenced by interfering substances which disrupt the binding between the analyte receptor and the analyte/labelled analyte. Also, the labelled analyte frequently will bind more strongly to the analyte receptor than the native analyte, adversely affecting the assay results.
Thus, it would be desirable to provide improved immunoassay formats which provide a visual signal which is related to a preselected concentration value of an analyte of interest. The visual signal should define a sharp end point, that is, should change from a minimum or zero value signal to a distinctly different signal over a very narrow concentration range centered at the preselected concentration value. The concentration value at which the visual signal changes should be independently selectable so that it can be correlated with a measurement value of interest. It would be further desirable if the selected concentration value could be programmed into the assay without the need to perform an additional step. Additionally, the assay of the present invention should overcome the disadvantages described above in connection with previous immunoassay systems.